In vitro development and metabolism of the human embryo up to the blastocyst stage

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P>0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P>0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

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35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab35 ◽

2011 ◽

Vol 23 (1) ◽

pp. 124

Author(s):

C. Feltrin ◽

M. Machado ◽

L. M. V. Queiroz ◽

M. A. S. Peixer ◽

P. F. Malard ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Zona Pellucida ◽

Blastocyst Stage ◽

Aggregation State ◽

Developmental Potential ◽

In Vitro Development ◽

Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

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Development of porcine oocytes from preovulatory follicles of different sizes after maturation in media supplemented with follicular fluids

Reproduction Fertility and Development ◽

10.1071/rd00027 ◽

2000 ◽

Vol 12 (4) ◽

pp. 133 ◽

Cited By ~ 60

Author(s):

Ki-Won Yoon ◽

Tae-Young Shin ◽

Jong-Im Park ◽

Sangho Roh ◽

Jeong M. Lim ◽

...

Keyword(s):

Blastocyst Stage ◽

Developmental Potential ◽

Large Follicle ◽

In Vitro Development ◽

Maturation Medium ◽

Preovulatory Follicles ◽

Vitro Development ◽

Small Follicle ◽

Follicular Fluids

The development of porcine oocytes from large (3.1–8.0 mm in diameter) or small (<3.1 mm) follicles was examined after maturation culture in medium containing porcine follicular fluid (pFF). Large follicles yielded larger (256 m v. 221 m; P<0.05) cumulus–oocyte complexes and more (22 v. 14%) morphologically normal oocytes than small follicles (Experiment 1). In Experiments 2–4, maturation media supplemented with mixed pFF (10%) from small and large follicles was used. More oocytes from large follicles matured (58% v. 91%), formed pronuclei (81% v. 90%) and developed to the blastocyst stage (2% v. 10%) than oocytes from small follicles. In Experiments 5–7, the effects of pFF collected from either small or large follicles on oocyte development were examined. Regardless of the source of oocytes, large-follicle-derived pFF more significantly enhanced preimplantation development than did small-follicle-derived pFF. The highest rate of blastocyst formation (16%) was found when oocytes from large follicles were cultured in maturation medium containing large-follicle-derived pFF. These results suggest that oocytes from large follicles have greater developmental potential than oocytes from small follicles, and that the origin of pFF, which is added to the maturation media, might be an important factor for improving in vitro development of porcine oocytes.

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43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH 6-DMAP FOLLOWING FUSION

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab43 ◽

2004 ◽

Vol 16 (2) ◽

pp. 144

Author(s):

G.-S. IM ◽

L. Lai ◽

Z. Liu ◽

Y. Hao ◽

C.M. Murphy ◽

...

Keyword(s):

Nuclear Transfer ◽

Electric Pulse ◽

Developmental Rate ◽

Cell Number ◽

Blastocyst Stage ◽

In Vitro Development ◽

Electric Pulses ◽

Developmental Rates ◽

Vitro Development

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P<0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P<0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.

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205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

Reproduction Fertility and Development ◽

10.1071/srb04abs205 ◽

2004 ◽

Vol 16 (9) ◽

pp. 205 ◽

Cited By ~ 2

Author(s):

K. M. Morton ◽

W. M. C. Maxwell ◽

G. Evans

Keyword(s):

Blastocyst Stage ◽

Developmental Competence ◽

Blastocyst Formation ◽

In Vitro Development ◽

Oocyte Collection ◽

Chi Squared ◽

Development In Vitro ◽

Stage 1 ◽

Vitro Development

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

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267 TARGETED SUPPRESSION OF THE EXPRESSION OF MATERNAL AND EMBRYONIC GENES DURING IN VITRO DEVELOPMENT OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab267 ◽

2006 ◽

Vol 18 (2) ◽

pp. 241

Author(s):

D. Tesfaye ◽

K. Nganvongpanit ◽

F. Rings ◽

M. Gilles ◽

D. Jennen ◽

...

Keyword(s):

Free Water ◽

Transcript Abundance ◽

Blastocyst Stage ◽

Bovine Embryos ◽

In Vitro Development ◽

First Polar Body ◽

Vitro Development ◽

Bovine Oocytes ◽

E Cadherin

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39�C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P < 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P < 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

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146 DETRIMENTAL EFFECTS OF PROSTAGLANDIN F2α ON IN VITRO EMBRYO DEVELOPMENT IN BOVINE ARE INHIBITED BY A RECEPTOR ANTAGONIST

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab146 ◽

2008 ◽

Vol 20 (1) ◽

pp. 153 ◽

Cited By ~ 1

Author(s):

F. N. Scenna ◽

J. L. Edwards ◽

F. N. Schrick

Keyword(s):

Prostaglandin F2α ◽

Blastocyst Stage ◽

Bovine Embryos ◽

Negative Effects ◽

In Vitro Development ◽

Ann Arbor ◽

Reproductive Techniques ◽

Vitro Development ◽

Cayman Chemical

Numerous studies have demonstrated negative effects of prostaglandin F2α (PGF2α) on bovine reproduction. Discovery of a PGF2α receptor (FPr) in bovine embryos (Scenna et al. 2006 Reprod. Fertil. Dev. 18, 180) allows for development of new therapeutic strategies to improve success of embryo transfer. Therefore, two experiments were performed to investigate the occurrence of any toxic effect of AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA), an FPr antagonist, on in vitro development of bovine embryos. In Exp. 1, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in potassium simplex optimized medium with polyvinyl alcohol (KSOM-PVA); n = 94); (2) 50 AL (50 nm AL-8810 in KSOM-PVA; n = 94); (3) 25 AL (25 nm AL-8810 in KSOM-PVA; n = 94); and (4) CON (control: KSOM-PVA; n = 95). In Exp. 2, pre-compacted embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 282); (2) 500 AL (500 nm AL-8810 in KSOM-PVA; n = 274); (3) 250 AL (250 nm AL-8810 in KSOM-PVA; n = 274); and (4) CON (control: KSOM-PVA; n = 278). Embryos remained in treatments until blastocyst assessment. Next, two experiments were performed to determine the efficiency of AL-8810 on preventing detrimental effects of PGF2α on pre-compacted embryos. In Exp. 3, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in KSOM-PVA; n = 121); (2) 10 PGF (10 ng mL–1 of PGF2α (Cayman Chemical Inc.) in KSOM-PVA; n = 91); (3) AL100+PGF (100 nm AL-8810 and 10 ng mL–1 of PGF2� in KSOM-PVA; n = 116); (4) CON (control: KSOM-PVA; n = 96). In Exp. 4, embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 87); (2) 10 PGF (10 ng mL–1 of PGF2α in KSOM-PVA; n = 87); (3) AL1000+PGF (1000 nm AL-8810 and 10 ng mL–1 of PGF2α in KSOM-PVA; n = 84); (4) CON (control: KSOM-PVA; n = 84). In Exp. 3 and 4, embryos remained in treatments for 48 h when development to morula was assessed. Data for all experiments were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). For Exp. 1, results indicated that addition of 100, 50, and 25 nm did not compromise embryonic development to the blastocyst stage compared to controls (60.2%, 55.8%, 55.4%, and 49.9%, respectively). In addition, orthogonal contrasts indicated that 100 nm AL-8810 improved development to the blastocyst stage (100 AL = 61% v. CON = 50.6%, P = 0.01). Similarly for Exp. 2, 1000, 500, and 250 nm AL-8810 did not affect in vitro development to the blastocyst stage compared to controls (40%, 39%, 34.8%, and 37.7%, respectively). In Exp. 3 and 4, addition of 1000 nm AL-8810, but not 100 nm, to culture medium of pre-compacted embryos exposed to PGF2α increased the ability of embryos to undergo compaction 48 h later (1000 AL+PGF = 51% v. PGF = 40%; P = 0.05). In conclusion, AL-8810 at a concentration of 1000 nm inhibits detrimental effects of PGF2α on the development of pre-compacted bovine embryos and may prove beneficial for other assisted reproductive techniques in cattle. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.

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26 EFFECT OF TREATMENT OF BOVINE DONOR CELLS WITH MOUSE EMBRYONIC STEM CELL EXTRACT ON THE DEVELOPMENT OF EMBRYOS AFTER NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab26 ◽

2011 ◽

Vol 23 (1) ◽

pp. 119

Author(s):

S. Akagi ◽

E. Mizutani ◽

Y. Inaba ◽

M. Kaneda ◽

T. Somfai ◽

...

Keyword(s):

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Cell Number ◽

Blastocyst Stage ◽

Control Group ◽

In Vitro Development ◽

Donor Cells ◽

Effect Of Treatment ◽

Vitro Development

The efficiency of somatic cell cloning is very low, probably because of incomplete reprogramming of the somatic cell nucleus. In recent studies, it is suggested that transient exposure of donor somatic cells to mouse embryonic stem cell (ESC) extract enhances pluripotency of the cells in vitro (Bru et al. 2008 Exp. Cell Res. 314, 1634–1642; Xu et al. 2009 Anat. Rec. 292, 1229–1234). In the present study, we examined the effect of treatment of donor cells with mouse ESC extract on the in vitro development of bovine NT embryos. First, in order to examine effect of treatment of donor cells with streptolysin O (SLO), which reversibly permeabilizes the plasma membrane, we compared the in vitro development of NT embryos using donor cells treated with 5 μg mL–1 SLO (SLO group) and untreated donor cells (control group). As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used. Fibroblasts were treated with 5 μg mL–1 SLO for 45 min, and then incubated for resealing in DMEM including 2 mM CaCl2 for 60 min. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). After in vitro culture for 8 days, blastocyst formation and cell number of blastocysts were examined. There were no significant differences between SLO and control groups in the fusion rate (80% and 72%, respectively), cleavage rate (60% and 65%, respectively), developmental rate to the blastocyst stage of NT embryos (31% and 28%, respectively), and blastocyst cell number (127 ± 6 and 112 ± 14, respectively). These results suggest that SLO treatment of donor cells has no negative effect on the in vitro development of NT embryos. Next, we examined the in vitro developmental ability of NT embryos using donor cells treated with mouse ESC extract (ES extract group). After SLO treatment for 45 min, permeabilized fibroblast cells were treated with mouse ESC extract for 45 min, and then incubated in DMEM including 2 mM CaCl2 for 60 min, and used for producing NT embryos. There were no differences between ES extract and control groups in the fusion rate (68% and 69%, respectively), cleavage rate (86.7% and 80.6%, respectively), and developmental rate to the blastocyst stage of NT embryos (39.8% and 43.5%, respectively). The cell number of NT embryos at the blastocyst stage in ES extract group (201 ± 30) was significantly (t-test; P < 0.05) higher than that in control group (140 ± 14). In conclusion, treatment of bovine donor cell with mouse ESC extract did not affect the in vitro developmental ability of NT embryos, but improved the quality of blastocysts.

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83 IN LOW OXYGEN CULTURE, IS HYPOTAURINE NECESSARY FOR IN VITRO DEVELOPMENT OF PORCINE EMBRYOS?

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab83 ◽

2016 ◽

Vol 28 (2) ◽

pp. 171

Author(s):

J. A. Benne ◽

L. D. Spate ◽

B. M. Elliott ◽

R. S. Prather

Keyword(s):

Embryo Culture ◽

Cell Number ◽

Blastocyst Stage ◽

Total Cell ◽

Free Radical Scavengers ◽

Total Cell Number ◽

In Vitro Development ◽

Significant Difference ◽

Vitro Development

For decades it has been known that reactive oxidative species (ROS) form during in vitro embryo culture. A buildup of ROS can be detrimental to individual cells in the embryo and lead to a decrease in development and quality. To overcome oxidative stress in culture systems, additives, such as taurine and/or hypotaurine, have been used. In the pig, taurine or hypotaurine addition is deemed necessary for normal in vitro development. Another commonly used technique to reduce ROS is to culture embryos in a lowered oxygen environment (e.g. 5%). Porcine zygote medium 3 (PZM3) base culture medium is used in the following experiments and contains 5 mM hypotaurine, which is one of the most costly additives in the medium. The objective of this experiment was to determine if hypotaurine is still necessary if the embryos were cultured in 5% O2 from the zygote to the Day 6 blastocyst stage. In Experiment 1, oocytes were matured for 44 h and fertilized in vitro. After fertilization, presumptive zygotes were then transferred to 500 µL of MU-1 medium (PZM3 with 1.69 mM arginine) that either contained or did not contain hypotaurine for overnight culture at 20% O2. On Day 1, the same embryo culture plates were moved to 5% O2, 5% CO2, and 90% N2 and cultured to Day 6. The percent blastocyst stage was determined, and total cell number was counted in 3 of the 5 replicates in order to give us an indication of the embryo quality. The percent blastocyst in the controls (+hypotaurine) was 34.4% ± 2.8 and not different from the no hypotaurine (32.9% ± 2.2; N = 830; 5 replications; P > 0.10). Furthermore, total cell number was not different between the two groups (30.8 ± 1.5 v. 33.6 ± 1.8, respectively, N = 146; 3 replications; P > 0.10). In Experiment 2, the same experiment was repeated in somatic cell nuclear transfer derived embryos, which may be more sensitive to ROS due to the micromanipulation procedure. Wild type fetal fibroblast cells were used as donor cells. There was no significant difference in development to the blastocyst stage due to the presence or absence of hypotaurine (17.7% ± 2.5 v. 11.8% ± 2.3, respectively; N = 454; 4 replications; P = 0.07). All blastocyst data were analysed using the GENMOD procedure in SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and cell number data were analysed using the PROC GLM also with SAS 9.4. These data show that porcine embryos can be efficiently cultured to the blastocyst stage without adding any oxygen free radical scavengers to the media when culturing in reduced oxygen atmosphere. Further studies include evaluating term development via embryo transfers and measuring ROS production of these embryos. Funding was provided by Food for the 21st Century and the National Institutes of Health (U42 OD011140).

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